Cultures of human CNS neural stem cells

ABSTRACT

Isolation, characterization, proliferation, differentiation and transplantation of mammalian neural stem cells is disclosed.

RELATED APPLICATIONS

This is a divisional application of U.S. patent application Ser. No.08/926,313, filed Sep. 5, 1997.

TECHNICAL FIELD OF THE INVENTION

This invention relates to isolation of human central nervous system stemcells, and methods and media for proliferating, differentiating andtransplanting them.

BACKGROUND OF THE INVENTION

During development of the central nervous system ("CNS"), multipotentprecursor cells, also known as neural stem cells, proliferate, givingrise to transiently dividing progenitor cells that eventuallydifferentiate into the cell types that compose the adult brain. Neuralstem cells are classically defined as having the ability to self-renew(i.e., form more stem cells), to proliferate, and to differentiate intomultiple different phenotypic lineages, including neurons, astrocytesand oligodendrocytes.

These neural stem cells have been isolated from several mammalianspecies, including mice, rats, pigs and humans. See, e.g., WO 93/01275,WO 94/09119, WO 94/10292, WO 94/16718 and Cattaneo et al., Mol. BrainRes., 42, pp. 161-66 (1996), all herein incorporated by reference.

Human CNS neural stem cells, like their rodent homologues, whenmaintained in a mitogen-containing (typically epidermal growth factor orepidermal growth factor plus basic fibroblast growth factor), serum-freeculture medium, grow in suspension culture to form aggregates of cellsknown as "neurospheres". In the prior art, human neural stem cells havedoubling rates of about 30 days. See, e.g., Cattaneo et al., Mol. BrainRes., 42, pp. 161-66 (1996). Upon removal of the mitogen(s) andprovision of a substrate, the stem cells differentiate into neurons,astrocytes and oligodendrocytes. In the prior art, the majority of cellsin the differentiated cell population have been identified asastrocytes, with very few neurons (<10%) being observed.

There remains a need to increase the rate of proliferation of neuralstem cell cultures. There also remains a need to increase the number ofneurons in the differentiated cell population. There further remains aneed to improve the viability of neural stem cell grafts uponimplantation into a host.

SUMMARY OF THE INVENTION

This invention provides novel human central nervous system stem cells,and methods and media for proliferating, differentiating andtransplanting them. In one embodiment, this invention provides novelhuman stem cells with a doubling rate of between 5-10 days, as well asdefined growth media for prolonged proliferation of human neural stemcells. The invention also provides differentiated cell populations ofhuman neural stem cells that provide previously unobtainable largenumbers of neurons, as well as astrocytes and oligodendrocytes. Thisinvention also provides novel methods for transplanting neural stemcells that improve the viability of the graft upon implantation in ahost.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representation of spheres of proliferating 9 FBr humanneural stem cells (passage 6) derived from human forebrain tissue.

FIG. 2, Panel A, shows a growth curve for a human neural stem cell linedesignated 6.5 Fbr cultured in (a) defined media containing EGF, FGF andleukemia inhibitory factor ("LIF") (shown as closed diamonds), and (b)the same media but without LIF (shown as open diamonds); Panel B shows agrowth curve for a human neural stem cell line designated 9 Fbr culturedin (a) defined media containing EGF, FGF and LIF (shown as closeddiamonds), and (b) the same media but without LIF (shown as opendiamonds); Panel C shows a growth curve for a human neural stem cellline designated 9.5 Fbr cultured in (a) defined media containing EGF,FGF and LIF (shown as closed diamonds), and (b) the same media butwithout LIF (shown as open diamonds); Panel D shows a growth curve for ahuman neural stem cell line designated 10.5 Fbr cultured in (a) definedmedia containing EGF, FGF and leukemia inhibitory factor ("LIF") (shownas closed diamonds), and (b) the same media but without LIF (shown asopen diamonds).

FIG. 3 shows a growth curve for a human neural stem cell line designated9 Fbr cultured in (a) defined media containing EGF and basic fibroblastgrowth factor ("bFGF") (shown as open diamonds), and (b) defined mediawith EGF but without bFGF (shown as closed diamonds).

FIG. 4 shows a graph of cell number versus days in culture for an Mx-1conditionally immortalized human glioblast line derived from a humanneural stem cell line. The open squares denote growth in the presence ofinterferon, the closed diamonds denote growth in the absence ofinterferon.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to isolation, characterization, proliferation,differentiation and transplantation of CNS neural stem cells.

In one embodiment the invention provides novel human CNS stem cellsisolated from the forebrain. We have isolated 4 neural stem cell linesfrom human forebrain, all of which exhibit neural stem cell properties;namely, the cells are self renewing, the cells proliferate for longperiods in mitogen containing serum free medium, and the cells, whendifferentiated, comprise a cell population of neurons, astrocytes andoligodendrocytes. These cells are capable of doubling every 5-10 days,in contrast with the prior art diencephalon-derived human neural stemcells. Reported proliferation rates of diencephalon-derived human neuralstem cells approximate one doubling every 30 days. See Cattaneo et al.,Mol. Brain Res., 42, pp. 161-66 (1996).

Any suitable tissue source may be used to derive the neural stem cellsof this invention. Both allografts and autografts are contemplated fortransplantation purposes.

This invention also provides a novel growth media for proliferation ofneural stem cells. Provided herein is a serum-free or serum-depletedculture medium for the short term and long term proliferation of neuralstem cells.

A number of serum-free or serum-depleted culture media have beendeveloped due to the undesirable effects of serum which can lead toinconsistent culturing results. See, e.g., WO 95/00632 (incorporatedherein by reference), and prior art medium described therein.

Prior to development of the novel media described herein, neural stemcells have been cultured in serum-free media containing epidermal growthfactor ("EGF") or an analog of EGF, such as amphiregulin or transforminggrowth factor alpha ("TGF-a"), as the mitogen for proliferation. See,e.g., WO 93/01275, WO 94/16718, both incorporated herein by reference.Further, basic fibroblast growth factor ("bFGF") has been used, eitheralone, or in combination with EGF, to enhance long term neural stem cellsurvival.

The improved medium according to this invention, which contains leukemiainhibitory factor ("LIF"), markedly and unexpectedly increases the rateof proliferation of neural stem cells, particularly human neural stemcells.

We have compared growth rates of the forebrain-derived stem cellsdescribed herein in the presence and absence of LIF; unexpectedly wehave found that LIF dramatically increases the rate of cellularproliferation in almost all cases.

The medium according to this invention comprises cell viability and cellproliferation effective amounts of the following components:

(a) a standard culture medium being serum-free (containing 0-0.49%serum) or serum-depleted (containing 0.5-5.0% serum), known as a"defined" culture medium, such as Iscove's modified DulbeccO's medium("IMDM"), RPMI, DMEM, Fischer's, alpha medium, Leibovitz's, L-15, NCTC,F-10, F-12, MEM and McCoy's;

(b) a suitable carbohydrate source, such as glucose;

(c) a buffer such as MOPS, HEPES or Tris, preferably HEPES;

(d) a source of hormones including insulin, transferrin, progesterone,selenium, and putrescine;

(e) one or more growth factors that stimulate proliferation of neuralstem cells, such as EGF, bFGF, PDGF, NGF, and analogs, derivativesand/or combinations thereof, preferably EGF and bFGF in combination;

(f) LIF

Standard culture media typically contains a variety of essentialcomponents required for cell viability, including inorganic salts,carbohydrates, hormones, essential amino acids, vitamins, and the like.We prefer DMEM or F-12 as the standard culture medium, most preferably a50/50 mixture of DMEM and F-12. Both media are commercially available(DMEM--Gibco 12100-046; F-12 - Gibco 21700-075). A premixed formulationis also commercially available (N2 - Gibco 17502-030). It isadvantageous to provide additional glutamine, preferably at about 2 mM.It is also advantageous to provide heparin in the culture medium.Preferably, the conditions for culturing should be as close tophysiological as possible. The pH of the culture medium is typicallybetween 6-8, preferably about 7, most preferably about 7.4. Cells aretypically cultured between 30-40° C., preferably between 32-38° C., mostpreferably between 35-37° C. Cells are preferably grown in 5% CO₂. Cellsare preferably grown in suspension culture.

In one exemplary embodiment, the neural stem cell culture comprises thefollowing components in the indicated concentrations:

    ______________________________________                                        Component        Final Concentration                                          ______________________________________                                        50/50 mix of DMEM/F-12                                                                         0.5-2.0 X, preferably 1X                                       glucose 0.2-1.0%, preferably 0.6% w/v                                         glutamine 0.1-10 mM, preferably 2 mM                                          NaHCO.sub.3 0.1-10 mM, preferably 3 mM                                        HEPES 0.1-10 mM, preferably 5 mM                                              apo-human transferrin (Sigma T- 1-1000 μg/ml, preferably 100                              μg/ml                                                       2252)                                                                         human insulin (Sigma I-2767) 1-100, preferably 25 μg/ml                    putrescine (Sigma P-7505) 1-500, preferably 60 μM                          selenium (Sigma S-9133) 1-100, preferably 30 nM                               progesterone (Sigma P-6149) 1-100, preferably 20 nM                           human EGF (Gibco 13247-010) 0.2-200, preferably 20 ng/ml                      human bFGF (Gibco 13256-029) 0.2-200, preferably 20 ng/ml                     human LIF (R&D Systems 250-L) 0.1-500, preferably 10 ng/ml                    heparin (Sigma H-3149) 0.1-50, preferably 2 μg/ml                          CO.sub.2 preferably 5%                                                      ______________________________________                                    

Serum albumin may also be present in the instant culturemedium--although the present medium is generally serum-depleted orserum-free (preferably serum-free), certain serum components which arechemically well defined and highly purified (>95%), such as serumalbumin, may be included.

The human neural stem cells described herein may be cryopreservedaccording to routine procedures. We prefer cryopreserving about one toten million cells in "freeze" medium which consists of proliferationmedium (absent the growth factor mitogens), 10% BSA (Sigma A3059) and7.5% DMSO. Cells are centrifuged. Growth medium is aspirated andreplaced with freeze medium. Cells are resuspended gently as spheres,not as dissociated cells. Cells are slowly frozen, by, e.g., placing ina container at -80° C. Cells are thawed by swirling in a 37° C. bath,resuspended in fresh proliferation medium, and grown as usual.

In another embodiment, this invention provides a differentiated cellculture containing previously unobtainable large numbers of neurons, aswell as astrocytes and oligodendrocytes. In the prior art, typically thedifferentiated human diencephalon-derived neural stem cell culturesformed very few neurons (i.e., less than 5-10%). According to thismethodology, we have routinely achieved neuron concentrations of between20% and 35% (and much higher in other cases) in differentiated humanforebrain-derived neural stem cell cultures. This is highly advantageousas it permits enrichment of the neuronal population prior toimplantation in the host in disease indications where neuronal functionhas been impaired or lost.

Further, according to the methods of this invention, we have achieveddifferentiated neural stem cell cultures that are highly enriched inGABA-ergic neurons. Such GABA-ergic neuron enriched cell cultures areparticularly advantageous in the potential therapy of excitotoxicneurodegenerative disorders, such as Huntington's disease or epilepsy.

In order to identify the cellular phenotype either during proliferationor differentiation of the neural stem cells, various cell surface orintracellular markers may be used.

When the neural stem cells of this invention are proliferating asneurospheres, we contemplate using human nestin antibody as a marker toidentify undifferentiated cells. The proliferating cells should showlittle GFAP staining and little b-tubulin staining (although somestaining might be present due to diversity of cells within the spheres).

When differentiated, most of the cells lose their nestin positiveimmunoreactivity. In particular, antibodies specific for variousneuronal or glial proteins may be employed to identify the phenotypicproperties of the differentiated cells. Neurons may be identified usingantibodies to neuron specific enolase ("NSE"), neurofilament, tau,b-tubulin, or other known neuronal markers. Astrocytes may be identifiedusing antibodies to glial fibrillary acidic protein ("GFAP"), or otherknown astrocytic markers. Oligodendrocytes may be identified usingantibodies to galactocerebroside, O4, myelin basic protein ("MBP") orother known oligodendrocytic markers.

It is also possible to identify cell phenotypes by identifying compoundscharacteristically produced by those phenotypes. For example, it ispossible to identify neurons by the production of neurotransmitters suchas acetylcholine, dopamine, epinephrine, norepinephrine, and the like.

Specific neuronal phenotypes can be identified according to the specificproducts produced by those neurons. For example, GABA-ergic neurons maybe identified by their production of glutamic acid decarboxylase ("GAD")or GABA. Dopaminergic neurons may be identified by their production ofdopa decarboxylase ("DDC"), dopamine or tyrosine hydroxylase ("TH").Cholinergic neurons may be identified by their production of cholineacetyltransferase ("ChAT"). Hippocampal neurons may be identified bystaining with NeuN. It will be appreciated that any suitable knownmarker for identifying specific neuronal phenotypes may be used.

The human neural stem cells described herein can be geneticallyengineered or modified according to known methodology.

A gene of interest (i.e., a gene that encodes a biologically activemolecule) can be inserted into a cloning site of a suitable expressionvector by using standard techniques. These techniques are well known tothose skilled in the art. See, e.g., WO 94/16718, incorporated herein byreference.

The expression vector containing the gene of interest may then be usedto transfect the desired cell line. Standard transfection techniquessuch as calcium phosphate co-precipitation, DEAE-dextran transfection,electroporation, biolistics, or viral transfection may be utilized.Commercially available mammalian transfection kits may be purchased frome.g., Stratagene. Human adenoviral transfection may be accomplished asdescribed in Berg et al. Exp. Cell Res., 192, pp. (1991). Similarly,lipofectamine-based transfection may be accomplished as described inCattaneo, Mol. Brain Res., 42, pp. 161-66 (1996).

A wide variety of host/expression vector combinations may be used toexpress a gene encoding a biologically active molecule of interest. See,e.g., U.S. Pat. No. 5,545,723, herein incorporated by reference, forsuitable cell-based production expression vectors.

Increased expression of the biologically active molecule can be achievedby increasing or amplifying the transgene copy number usingamplification methods well known in the art. Such amplification methodsinclude, e.g., DHFR amplification (see, e.g., Kaufman et al., U.S. Pat.No. 4,470,461) or glutamine synthetase ("GS") amplification (see, e.g.,U.S. Pat. No. 5,122,464, and European published application EP 338,841),all herein incorporated by reference.

The neural stem cells described herein, and their differentiated progenymay be immortalized or conditionally immortalized using knowntechniques. We prefer conditional immortalization of stem cells, andmost preferably conditional immortalization of their differentiatedprogeny. Among the conditional immortalization techniques contemplatedare Tet-conditional immortalization (see WO 96/31242, incorporatedherein by reference), and Mx-1 conditional immortalization (see WO96/02646, incorporated herein by reference).

This invention also provides methods for differentiating neural stemcells to yield cell cultures enriched with neurons to a degreepreviously unobtainable. According to one protocol, the proliferatingneurospheres are induced to differentiate by removal of the growthfactor mitogens and LIF, and provision of 1% serum, a substrate and asource of ionic charges (e.g., glass cover slip covered withpoly-ornithine or extracellular matrix components). The preferred basemedium for this differentiation protocol, excepting the growth factormitogens and LIF, is otherwise the same as the proliferation medium.This differentiation protocol produces a cell culture enriched inneurons. According to this protocol, we have routinely achieved neuronconcentrations of between 20% and 35% in differentiated humanforebrain-derived neural stem cell cultures.

According to a second protocol, the proliferating neurospheres areinduced to differentiate by removal of the growth factor mitogens, andprovision of 1% serum, a substrate and a source of ionic charges (e.g.,glass cover slip covered with poly-ornithine or extracellular matrixcomponents), as well as a mixture of growth factors including PDGF,CNTF, IGF-1, LIF, forskolin, T-3 and NT-3. The cocktail of growthfactors may be added at the same time as the neurospheres are removedfrom the proliferation medium, or may be added to the proliferationmedium and the cells pre-incubated with the mixture prior to removalfrom the mitogens. This protocol produces a cell culture highly enrichedin neurons and enriched in oligodendrocytes. According to this protocol,we have routinely achieved neuron concentrations of higher than 35% indifferentiated human forebrain-derived neural stem cell cultures.

The human stem cells of this invention have numerous uses, including fordrug screening, diagnostics, genomics and transplantation.

The cells of this invention may be transplanted "naked" into patientsaccording to conventional techniques, into the CNS, as described forexample, in U.S. Pat. Nos. 5,082,670 and 5,618,531, each incorporatedherein by reference, or into any other suitable site in the body.

In one embodiment, the human stem cells are transplanted directly intothe CNS. Parenchymal and intrathecal sites are contemplated. It will beappreciated that the exact location in the CNS will vary according tothe disease state. According to one aspect of this invention, providedherein is methodology for improving the viability of transplanted humanneural stem cells. In particular, we have discovered that graftviability improves if the transplanted neural stem cells are allowed toaggregate, or to form neurospheres prior to implantation, as compared totransplantation of dissociated single cell suspensions. We prefertransplanting small sized neurospheres, approximately 10-500 μm indiameter, preferably 40-50 μm in diameter. Alternatively, we preferspheres containing about 5-100, preferably 5-20 cells per sphere. Wecontemplate transplanting at a density of about 10,000-1,000,000 cellsper μl, preferably 25,000-500,000 cells per μl.

The cells may also be encapsulated and used to deliver biologicallyactive molecules, according to known encapsulation technologies,including microencapsulation (see, e.g., U.S. Pat. Nos. 4,352,883;4,353,888; and 5,084,350, herein incorporated by reference), (b)macroencapsulation (see, e.g., U.S. Pat. Nos. 5,284,761, 5,158,881,4,976,859 and 4,968,733 and published PCT patent applicationsW092/19195, WO 95/05452, each incorporated herein by reference).

If the human neural stem cells are encapsulated, we prefermacroencapsulation, as described in U.S. Pat. Nos. 5,284,761; 5,158,881;4,976,859; 4,968,733; 5,800,828 and published PCT patent application WO95/05452, each incorporated herein by reference. Cell number in thedevices can be varied; preferably each device contains between 10³ -10⁹cells, most preferably 10⁵ to 10⁷ cells. A large number ofmacroencapsulation devices may be implanted in the patient; we preferbetween one to 10 devices.

In addition, we also contemplate "naked" transplantation of human stemcells in combination with a capsular device wherein the capsular devicesecretes a biologically active molecule that is therapeuticallyeffective in the patient or that produces a biologically active moleculethat has a growth or trophic effect on the transplanted neural stemcells, or that induces differentiation of the neural stem cells towardsa particular phenotypic lineage.

The cells and methods of this invention may be useful in the treatmentof various neurodegenerative diseases and other disorders. It iscontemplated that the cells will replace diseased, damaged or losttissue in the host. Alternatively, the transplanted tissue may augmentthe function of the endogenous affected host tissue. The transplantedneural stem cells may also be genetically modified to provide abiologically active molecule that is therapeutically effective.

Excitotoxicity has been implicated in a variety of pathologicalconditions including epilepsy, stroke, ischemia, and neurodegenerativediseases such as Huntington's disease, Parkinson's disease andAlzheimer's disease. Accordingly, neural stem cells may provide onemeans of preventing or replacing the cell loss and associated behavioralabnormalities of these disorders.

Huntington's disease (HD) is an autosomal dominant neurodegenerativedisease characterized by a relentlessly progressive movement disorderwith devastating psychiatric and cognitive deterioration. HD isassociated with a consistent and severe atrophy of the neostriatum whichis related to a marked loss of the GABAergic medium-sized spinyprojection neurons, the major output neurons of the striatum.Intrastriatal injections of excitotoxins such as quinolinic acid (QA)mimic the pattern of selective neuronal vulnerability seen in HD. QAlesions result in motor and cognitive deficits which are among the majorsymptoms seen in HD. Thus, intrastriatal injections of QA have become auseful model of HD and can serve to evaluate novel therapeuticstrategies aimed at preventing, attenuating, or reversingneuroanatomical and behavioral changes associated with HD. BecauseGABA-ergic neurons are characteristically lost in Huntington's disease,we contemplate treatment of Huntington's patients by transplantation ofcell cultures enriched in GABA-ergic neurons derived according to themethods of this invention.

Epilepsy is also associated with excitotoxicity. Accordingly, GABA-ergicneurons derived according to this invention are contemplated fortransplantation into patients suffering from epilepsy.

We also contemplate use of the cells of this invention in the treatmentof various demyelinating and dysmyelinating disorders, such asPelizaeus-Merzbacher disease, multiple sclerosis, variousleukodystrophies, post-traumatic demyelination, and cerebrovascular(CVS) accidents, as well as various neuritis and neuropathies,particularly of the eye. We contemplate using cell cultures enriched inoligodendrocytes or oligodendrocyte precursor or progenitors, suchcultures prepared and transplanted according to this invention topromote remyelination of demyelinated areas in the host.

We also contemplate use of the cells of this invention in the treatmentof various acute and chronic pains, as well as for certain nerveregeneration applications (such as spinal cord injury). We alsocontemplate use of human stem cells for use in sparing or sprouting ofphotoreceptors in the eye.

The cells and methods of this invention are intended for use in amammalian host, recipient, patient, subject or individual, preferably aprimate, most preferably a human.

The following examples are provided for illustrative purposes only, andare not intended to be limiting.

EXAMPLES Example 1 Media for Proliferating Neural Stem Cells

Proliferation medium was prepared with the following components in theindicated concentrations:

    ______________________________________                                        Component            Final Concentration                                      ______________________________________                                        50/50 mix of DMEM/F-12                                                                             1X                                                       glucose              0.6%      w/v                                              glutamine 2 mM                                                                NaHCO.sub.3 3 mM                                                              HEPES 5 mM                                                                    apo-human transferrin (Sigma T-2252) 100 μg/ml                             human insulin (Sigma I-2767) 25 μg/ml                                      putrescine (Sigma P-7505) 60 μM                                            selenium (Sigma S-9133) 30 nM                                                 progesterone (Sigma P-6149) 20 nM                                             human EGF (Gibco 13247-010) 20 ng/ml                                          human bFGF (Gibco 13256-029) 20 ng/ml                                         human LIF (R&D Systems 250-L) 10 ng/ml                                        heparin (Sigma H-3149) 2 μg/ml                                           ______________________________________                                    

Example 2 Isolation of Human CNS Neural Stem Cells

Sample tissue from human embryonic forebrain was collected and dissectedin Sweden and kindly provided by Huddinje Sjukhus. Blood samples fromthe donors were sent for viral testing. Dissections were performed insaline and the selected tissue was placed directly into proliferationmedium (as described in Example 1). Tissue was stored at 4° C. untildissociated. The tissue was dissociated using a standard glasshomogenizer, without the presence of any digesting enzymes. Thedissociated cells were counted and seeded into flasks containingproliferation medium. After 5-7 days, the contents of the flasks arecentrifuged at 1000 rpm for 2 min. The supernatant was aspirated and thepellet resuspended in 200 μl of proliferation medium. The cell clusterswere triturated using a P200 pipetman about 100 times to break up theclusters. Cells were reseeded at 75,000-100,000 cells/ml intoproliferation medium. Cells were passaged every 6-21 days depending uponthe mitogens used and the seeding density. Typically these cellsincorporate BrdU, indicative of cell proliferation. For T75 flaskcultures (initial volume 20 ml), cells are "fed" 3 times weekly byaddition of 5 ml of proliferation medium. We prefer Nunc flasks forculturing.

Nestin Staining for Proliferating Neurospheres

We stained for nestin (a measure of proliferating neurospheres) asfollows. Cells were fixed for 20 min at room temperature with 4%paraformaldehyde. Cells were washed twice for 5 min with 0.1 M PBS, pH7.4. Cells were permeabilized for 2 min with 100% EtOH. The cells werethen washed twice for 5 min with 0.1 M PBS. Cell preparations wereblocked for 1 hr at room temperature in 5% normal goat serum ("NGS")diluted in 0.1 M PBS, pH 7.4 and 1% Triton X-100 (Sigma X-100) for 1 hrat room temperature with gentle shaking. Cells were incubated withprimary antibodies to human nestin (from Dr. Lars Wahlberg, Karolinska,Sweden, rabbit polyclonal used at 1:500) diluted in 1% NGS and 1% TritonX-100 for 2 hr at room temperature. Preparations were then washed twicefor 5 min with 0.1 M PBS. Cells were incubated with secondary antibodies(pool of GAM/FITC used at 1:128, Sigma F-0257; GAR/TRITC used at 1:80,Sigma T-5268) diluted in 1% NGS and 1% Triton X-100 for 30 min at roomtemperature in the dark. Preparations are washed twice for 5 min with0.1 M PBS in the dark. Preparations are mounted onto slides face downwith mounting medium (Vectashield Mounting Medium, Vector Labs., H-1000)and stored at 4° C.

FIG. 1 shows a picture of proliferating spheres (here called"neurospheres") of human forebrain derived neural stem cells. Weevaluated proliferation of 4 lines of human forebrain derived neuralstem cells in proliferation medium as described above with LIF presentof absent.

As FIG. 2 shows, in three of the four lines (6.5 Fbr, 9 Fbr, and 10.5FBr), LIF significantly increased the rate of cell proliferation. Theeffect of LIF was most pronounced after about 60 days in vitro.

We also evaluated the effect of bFGF on the rate of proliferation ofhuman forebrain-derived neural stem cells. As FIG. 3 shows, in thepresence of bFGF, the stem cells proliferation was significantlyenhanced.

Example 3 Differentiation of Human Neural Stem Cells

In a first differentiation protocol, the proliferating neurospheres wereinduced to differentiate by removal of the growth factor mitogens andLIF, and provision of 1% serum, a substrate and a source of ioniccharges(e.g., glass cover slip covered with poly-ornithine).

The staining protocol for neurons, astrocytes and oligodendrocytes wasas follows:

β-Tubulin Staining for Neurons

Cells were fixed for 20 min at room temperature with 4%paraformaldehyde. Cells were washed twice for 5 min with 0.1 M PBS, pH7.4. Cells were permeabilized for 2 min with 100% EtOH. The cells werethen washed twice for 5 min with 0.1 M PBS. Cell preparations wereblocked for 1 hr at room temperature in 5% normal goat serum ("NGS")diluted in 0.1 M PBS, pH 7.4. Cells were incubated with primaryantibodies to b-tubulin (Sigma T-8660, mouse monoclonal; used at 1:1,000) diluted in 1% NGS for 2 hr at room temperature. Preparations werethen washed twice for 5 min with 0.1 M PBS. Cells were incubated withsecondary antibodies (pool of GAM/FITC used at 1:128, Sigma F-0257;GAR/TRITC used at 1:80, Sigma T-5268) diluted in 1% NGS for 30 min atroom temperature in the dark. Preparations are washed twice for 5 minwith 0.1 M PBS in the dark. Preparations are mounted onto slides facedown with mounting medium (Vectashield Mounting Medium, Vector Labs.,H1000) and stored at 4° C.

In some instances we also stain with DAPI (a nuclear stain), as follows.Coverslips prepared as above are washed with DAPI solution (diluted1:1000 in 100% MeOH, Boehringer Mannheim, # 236 276). Coverslips areincubated in DAPI solution for 15 min at 37° C.

O4 Staining for Oligodendrocvtes

Cells were fixed for 10 min at room temperature with 4%paraformaldehyde. Cells were washed three times for 5 min with 0.1 MPBS, pH 7.4. Cell preparations were blocked for 1 hr at room temperaturein 5% normal goat serum ("NGS") diluted in 0.1 M PBS, pH 7.4. Cells wereincubated with primary antibodies to O4 (Boehringer Mannheim # 1518 925,mouse monoclonal; used at 1:25) diluted in 1% NGS for 2 hr at roomtemperature. Preparations were then washed twice for 5 min with 0.1 MPBS. Cells were incubated with secondary antibodies, and furtherprocessed as described above for b-tubulin.

GFAP Staining for Astrocytes

Cells were fixed for 20 min at room temperature with 4%paraformaldehyde. Cells were washed twice for 5 min with 0.1 M PBS, pH7.4. Cells were permeabilized for 2 min with 100% EtOH. The cells werethen washed twice for 5 min with 0.1 M PBS. Cell preparations wereblocked for 1 hr at room temperature in 5% normal goat serum ("NGS")diluted in 0.1 M PBS, pH 7.4. Cells were incubated with primaryantibodies to GFAP (DAKO Z 334, rabbit polyclonal; used at 1:500)diluted in 1% NGS for 2 hr at room temperature. Preparations were thenwashed twice for 5 min with 0.1 M PBS. Cells were incubated withsecondary antibodies, and further processed as described above forb-tubulin.

This differentiation protocol produced cell cultures enriched in neuronsas follows:

    ______________________________________                                                         % GFAP   % b-tubulin                                                                           % of neurons that                             Cell Line Passage Positive positive are GABA positive                       ______________________________________                                        6.5  FBr    5        15     37      20                                          9 FBr 7 52 20 35                                                              10.5 FBr 5 50 28 50                                                         ______________________________________                                    

We also evaluated the ability of a single cell line to differentiateconsistently as the culture aged (i.e., at different passages), usingthe above differentiation protocol. The data are as follows:

    ______________________________________                                                        % GFAP    % b-tubulin                                                                           % of neurons that                             Cell Line Passage Positive positive are GABA positive                       ______________________________________                                        9 FBr  7        53        20.4    ND                                            9 FBr 9 ND 20.3 34.5                                                          9 FBr 15   62 17.9 37.9                                                     ______________________________________                                    

We conclude from these data that cells will follow reproducibledifferentiation patterns irrespective of passage number or culture age.

Example 4 Differentiation of Human Neural Stem Cells

In a second differentiation protocol, the proliferating neurosphereswere induced to differentiate by removal of the growth factor mitogensand LIF, and provision of 1% serum, a substrate (e.g., glass cover slipor extracellular matrix components), a source of ionic charges (e.g.,poly-ornithine) as well as a mixture of growth factors including 10ng/ml PDGF A/B, 10 ng/ml CNTF, 10 ng/ml IGF-1, 10 μM forskolin, 30 ng/mlT3, 10 ng/ml LIF and 1 ng/ml NT-3. This differentiation protocolproduced cell cultures highly enriched in neurons (i.e., greater than35% of the differentiated cell culture) and enriched inoligodendrocytes.

Example 5 Genetic Modification

We have conditionally immortalized a glioblast cell line derived fromthe human neural stem cells described herein, using the Mx-1 systemdescribed in WO 96/02646. In the Mx-1 system, the Mx-1 promoter drivesexpression of the SV40 large T antigen. The Mx-1 promoter is induced byinterferon. When induced, large T is expressed, and quiescent cellsproliferate.

Human glioblasts were derived from human forebrain neural stem cells asfollows. Proliferating human neurospheres were removed fromproliferation medium and plated onto poly-ornithine plastic (24 wellplate) in a mixture of N2 with the mitogens EGF, bFGF and LIF, as wellas 0.5% FBS. 0.5 ml of N2 medium and 1% FBS was added. The cells wereincubated overnight. The cells were then transfected with p318 (aplasmid containing the Mx-1 promoter operably linked to the SV 40 largeT antigen) using Invitrogen lipid kit (lipids 4 and 6). The transfectionsolution contained 6 μI/ml of lipid and 4 μl/ml DNA in optiMEM medium.The cells were incubated in transfection solution for 5 hours. Thetransfection solution was removed and cells placed into N2 and 1% FBSand 500 U/ml A/D interferon. The cells were fed twice a week. After tenweeks cells were assayed for large T antigen expression. The cellsshowed robust T antigen staining at this time. As FIG. 4 shows, cellnumber was higher in the presence of interferon than in the absence ofinterferon.

Large T expression was monitored using immunocytochemistry as follows.Cells were fixed for 20 min at room temperature with 4%paraformaldehyde. Cells were washed twice for 5 min with 0.1 M PBS, pH7.4. Cells were permeabilized for 2 min with 100% EtOH. The cells werethen washed twice for 5 min with 0.1 M PBS. Cell preparations wereblocked for 1 hr at room temperature in 5% normal goat serum ("NGS")diluted in 0.1 M PBS, pH 7.4. Cells were incubated with primaryantibodies to large T antigen (used at 1:10) diluted in 1% NGS for 2 hrat room temperature. We prepared antibody to large T antigen in house byculturing PAB 149 cells and obtaining the conditioned medium.Preparations were then washed twice for 5 min with 0.1 M PBS. Cells wereincubated with secondary antibodies (goat-anti-mouse biotinylated at1:500 from Vector Laboratories, Vectastain Elite ABC mouse IgG kit,PK-6102) diluted in 1% NGS for 30 min at room temperature. Preparationsare washed twice for 5 min with 0.1 M PBS. Preparations are incubated inABC reagent diluted 1:500 in 0.1 M PBS, pH 7.4 for 30 min at roomtemperature. Cells are washed twice for 5 min in 0.1 M PBS, pH 7.4, thenwashed twice for 5 min in 0.1 M Tris, pH 7.6. Cells are incubated in DAB(nickel intensification) for 5 min at room temperature. The DAB solutionis removed, and cells are washed three to five times with dH20. Cellsare stored in 50% glycerol/50% 0.1 M PBS, pH 7.4.

Example 6 Encapsulation

If the human neural stem cells are encapsulated, then the followingprocedure may be used:

The hollow fibers are fabricated from a polyether sulfone (PES) with anoutside diameter of 720 m and a wall thickness of a 100 m (AKZO-NobelWuppertal, Germany). These fibers are described in U.S. Pat. Nos.4,976,859 and 4,968,733, herein incorporated by reference. The fiber maybe chosen for its molecular weight cutoff. We sometimes use a PES#5membrane which has a MWCO of about 280 kd. In other studies we use aPES#8 membrane which has a MWCO of about 90 kd.

The devices typically comprise:

1) a semipermeable poly (ether sulfone) hollow fiber membrane fabricatedby AKZO Nobel Faser AG;

2) a hub membrane segment;

3) a light cured methacrylate (LCM) resin leading end; and

4) a silicone tether.

The semipermeable membrane used typically has the followingcharacteristics:

    ______________________________________                                        Internal Diameter 500 + 30 m                                                    Wall Thickness 100 + 15 m                                                     Force at Break 100 + 15 cN                                                    Elongation at Break 44 + 10%                                                  Hydraulic Permeability 63 + 8 (ml/min m.sup.2 mmHg)                           nMWCO (dextrans) 280 + 20 kd                                                ______________________________________                                    

The components of the device are commercially available. The LCM glue isavailable from Ablestik Laboratories (Newark, Del.); Luxtrak AdhesivesLCM23 and LCM24). The tether material is available from SpecialtySilicone Fabricators (Robles, Calif.). The tether dimensions are 0.79 mmOD×0.43 mm ID×length 202 mm. The morphology of the device is as follows:The inner surface has a permselective skin. The wall has an open cellfoam structure. The outer surface has an open structure, with pores upto 1.5 m occupying 30+5% of the outer surface.

Fiber material is first cut into 5 cm long segments and the distalextremity of each segment sealed with a photopolymerized acrylic glue(LCM-25, ICI). Following sterilization with ethylene oxide andoutgassing, the fiber segments are loaded with a suspension of between10⁴ -10⁷ cells, either in a liquid medium, or a hydrogel matrix (e.g., acollagen solution (Zyderm®), alginate, agarose or chitosan) via aHamilton syringe and a 25 gauge needle through an attached injectionport. The proximal end of the capsule is sealed with the same acrylicglue. The volume of the device contemplated in the human studies isapproximately 15-18 1.

A silicone tether (Specialty Silicone Fabrication, Taunton, Mass.) (ID:690 m; OD: 1.25 mm) is placed over the proximal end of the fiberallowing easy manipulation and retrieval of the device.

Example 8 Transplantation of Neural Stem Cells

We have transplanted human neural stem cells into rat brain and assessedgraft viability, integration, phenotypic fate of the grafted cells, aswell as behavioral changes associated with the grafted cells in lesionedanimals.

Transplantation was performed according to standard techniques. Adultrats were anesthetized with sodium pentobarbitol (45 mg/kg, i.p.) Andpositioned in a Kopf stereotaxic instrument. A midline incision was madein the scalp and a hole drilled for the injection of cells. Ratsreceived implants of unmodified, undifferentiated human neural stemcells into the left striatum using a glass capillary attached to a 10 μlHamilton syringe. Each animal received a total of about 250,000-500,000cells in a total volume of 2 μl. Cells were transplanted 1-2 days afterpassaging and the cell suspension was made up of undifferentiated stemcell clusters of 5-20 cells. Following implantation, the skin wassutured closed.

Animals were behaviorally tested and then sacrificed for histologicalanalysis.

I claim:
 1. A culture medium for proliferating mammalian neural stemcells, the media comprising cell viability and cell proliferationeffective amounts of the following components:(a) a standard definedbasal medium; (b) a supplemental carbohydrate, wherein the carbohydrateis present at a concentration between 0.2% and 1.0% weight/volume (w/v);(c) a supplemental buffer, such that the pH of the culture medium isbetween pH 6 and pH 8; (d) supplemental agents, wherein the agents areselected from the group consisting of insulin, transferrin,progesterone, selenium, putrescine, and combinations thereof; (e) one ormore growth factors that stimulate proliferation of neural stem cells,wherein the growth factors are present at a concentration of between 0.2ng/ml and 200 ng/ml, and; (f) leukocyte inhibitory factor (LIF), whereinthe LIF is present at a concentration of between 0.1 ng/ml and 500ng/ml.
 2. The medium of claim 1 further comprising heparin.